Probe carrier, probe fixing carrier and method of manufacturing the same

ABSTRACT

A carrier having indexes in a probe non-fixing region is used and solutions containing probes are applied to respective specific positions on the carrier by referring to said indexes and fixed. The position of a target compound that is specifically bonded to a probe fixed to a probe carrier manufactured by a method according to the invention can be accurately and quickly detected by referring to the indexes.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to a method of manufacturing a probe carrier byapplying a probe solution to a specific position on a carrier, utilizingone or more than one indexes and an ink-jet method in particular. Italso relates to a probe carrier manufactured by such a manufacturingmethod and a method of identifying the position of the target substancebonded to the probe on such a probe carrier by utilizing such indexes.

2. Related Background Art

When analyzing the base sequence of a gene DNA or conducting a genediagnosis for a number of items simultaneously, probes of differenttypes are needed to single out a DNA having a target base sequence inorder to raise the reliability of operation. DNA microchips have beenattracting attention as means for providing probes of a number ofdifferent types to be used for such sorting operations. A large numberof solution specimens (e.g., 96, 384 or 1,536 specimens) containingproteins or drugs to be sorted normally have to be subjected to ascreening operation in an orderly manner in the field of high throughputscreening of chemicals or combinatorial chemistry. For these purposes,techniques of sequentially arranging a large number of different typesof drugs, automatic screening technologies and dedicated devices forsorting the drugs arranged in this way and software for controlling anumber of screening operations and statistically processing the obtainedresults have been and being developed.

Basically, such screening operations as described above that areconducted in parallel simultaneously consist in detecting an action ornon-action or a response or non-response of each specimen to the knownprobes arranged in array, or probe array, provided as means for sortingthe substances of specimens for evaluation under same conditions.Generally, the action or response to be used with each probe is definedin advance and therefore substances (drugs) of a same type are normallyused as probe species that are mounted on a probe array. Then, the probearray may be that of DNA probes carrying a group of DNAs havingdifferent respective base sequences. DNAs, proteins and synthesizedchemicals are examples of substances that may be used for a group ofprobes. While a probe array of a group of a plurality of probe speciesis used in many instances, a large number of identical DNAs having asame base sequence, identical proteins having a same amino acid sequenceor identical chemical substances may be arranged in array depending onthe type of screening operation. Such probes are mainly used forscreening drugs.

In a probe array formed by a group of a plurality of probe species, aplurality of species of a group of DNAs having different base sequences,a group of proteins having different amino acid sequences, a group ofdifferent chemical substances or the like are often arranged in array ona substrate according to a predetermined order of arrangement.Particularly, DNA probe arrays are used for analyzing the base sequenceof a gene DNA or conducting a gene diagnosis by analyzing a number ofitems simultaneously in order to raise the reliability of operation aspointed out above.

One of the problems to be solved for probe arrays formed by a group of aplurality of probe species is how to mount as many probes of differentspecies, DNA probes having different base sequences for example, aspossible on a single carrier. Differently stated, it is a problem of howto mount probes in array as densely as possible.

U.S. Pat. No. 5,424,186 describes a technique of preparing an array ofDNA probes having respective base sequences that are different from eachother by means of a stepwise elongation reaction of DNAs conducted on acarrier by utilizing photodecomposable protective groups andphotolithography. With the proposed technique, it is possible to preparea DNA probe array carrying DNAs of more than 10,000 different kinds thatare different from each other in terms of base sequence per 1 cm². Theprocess of synthesizing a DNA by means of a stepwise elongationreaction, using this technique, comprises a photolithography step inwhich dedicated photomasks are used respectively for the four differentkinds of base (A, T, C, G) in order to selectively elongate any of thebases at a predetermined position of the array so that consequently DNAsof different species having desired respective base sequences aresynthetically produced and arranged on a substrate in a predeterminedorder. Then, the cost and the time required for preparing such a probearray rise as the DNA chain length increases. Furthermore, since theefficiency of nucleotide synthesis is not 100%, the ratio of DNAs thatare defective in terms of the designed base sequence is not negligible.Additionally, when photodecomposable protective groups are used for thesynthesis process, the efficiency of synthesis is rather poor ifcompared with the use of ordinary acid-decomposable protective groups.Therefore, the ratio of the DNAs that show the designed respective basesequences in the ultimately obtained array can be relatively small.

Besides, with the above identified known technique, since the productsformed synthetically and directly on a carrier have to be used withoutany modification, it is not possible to sort out the DNAs having adefective base sequence from the DNAs having the designed respectivebase sequences and eliminate the former for the purpose of refining.There is also a problem that it is not possible to confirm the basesequences of the DNAs synthetically formed on the carrier and ultimatelyobtained as an array. This means that, if a base has not been subjectedto predetermined elongation in a given elongation step probably becauseof an error or another in the step and hence the obtained probe array isnot good, any screening operation using such a defective probe arraygives rise to false results but there is no way of preventing such aproblem from taking place. In short, absence of confirmation of basesequences is the largest and most intrinsic problem of the aboveidentified known technique.

Apart from the above technique, techniques of manufacturing a probearray by synthesizing DNAs for probes in advance in a refined manner,confirming, if necessary, their respective base lengths and applying theDNAs to a carrier by means of an appropriate device such as amicrodispenser are also known. PCT Patent Publication WO95/35505describes a technique of applying DNAs onto a membrane by means ofcapillaries. With this technique, it is theoretically possible toprepare a DNA array having about 1,000 DNAs per 1 cm². It is basically atechnique of preparing a probe array by applying a probe solution to apredetermined position of a carrier for each probe by means of a singlecapillary-shaped dispensing device and repeating this operation. Whileno problem may arise when each probe is applied with a dedicatedcapillary-shaped dispensing device, a mutual contamination problem willoccur if a small number of capillary-shaped dispensing devices are usedrepeatedly for the operation, so that the capillary-shaped dispensingdevices have to be cleaned sufficiently each time a new probe species isbrought in to avoid such a mutual contamination problem. Additionally,the position where each probe solution is applied needs to be controlledaccurately. Therefore, this technique is not suited for preparing aprobe array comprising a wide variety of probes that are arrangeddensely. Still additionally, the operation of applying a probe solutionto the carrier is conducted by tapping the capillary tip to the carrierand hence not satisfactory in terms of both reproducibility andreliability.

There are also known techniques of applying a solution of a substancenecessary for conducting an operation of DNA solid phase synthesis on asubstrate in each elongation step by utilizing an ink-jet method. Forexample, European Patent Publication EP 0 703 825 B1 describes atechnique of synthesizing DNAs of a plurality of different specieshaving respective predetermined base sequences in a solid phase byapplying nucleotide monomers and activators by means of respective piezojet nozzles for the purpose of solid phase synthesis of DNAs. Thisapplication technique utilizing an ink-jet method is reliable in termsof reproducibility of the rate of application if compared with asolution application technique utilizing capillaries and also providesadvantages for realizing high density probe arrays because the nozzlestructure of the ink-jet system can be miniaturized. However, thistechnique is basically an application of a stepwise elongation reactionon a carrier and hence is not free from certain problems including thatof being unable to confirm the base sequences of the DNAs syntheticallyformed on the carrier as pointed out earlier to be the largest problemor the technique according to U.S. Pat. No. 5,424,186. While the problemof conducting a cumbersome photolithography operation, using a dedicatedmask in each elongation step, is dissolved with this technique, thistechnique is still accompanied to a certain extent by problems in termsof fixing predefined probes at respective positions, which is therequirement to be indispensably met for forming a probe array. It shouldbe noted here that the above cited EP 0 703 825 B1 only describes amethod of using a plurality of piezoelectric jet nozzles that are formedindependently. The use of a small number of such nozzles is not suitedfor preparing high density probe arrays like the above described methodof using capillary-shaped dispensing devices.

Japanese Patent Application Laid-open No. 11-187900 discloses a methodof forming spots containing probes on a solid phase by causing dropletsof probe-containing liquid to adhere to the solid phase by means ofthermal ink-jet heads.

When preparing a probe array by utilizing an ink-jet method, it isdesired from the viewpoint of high density arrangement to fix probes ofas many different types as possible within a given area in order toimprove the detection efficiency of diagnostic operations and avoid theneed of preparing specimens to a large quantity. Additionally, it isnecessary to accurately apply specific probes to respective intendedpositions from the viewpoint of reliability. It is also desired thatonly a right probe is fixed at an intended position from the viewpointof eliminating diagnosis errors.

As pointed out above, more accurate position control will be required inthe near future for fixing probes highly densely. However, with knownprobe fixing methods that utilize a conventional ink-jet process, it isoften impossible to apply liquid accurately to a desired position as theliquid ejecting operation is conducted by regulating the relativepositions of the carrier and the ink-jet head, visually confirming theposture of the entire carrier.

Additionally, when manufacturing a probe carrier by ejecting liquid in anumber of times, using an ink-jet method, the carrier may have to bealigned with the ink-jet head for each liquid-ejecting operation. Then,when a liquid-ejecting operation is concluded, the entire probe formedby applying the liquid is positionally checked before the nextliquid-ejecting operation is conducted. However, if the probe solutionapplied to the carrier by the last liquid-ejecting operation dries, itwill no longer be possible to visually ascertain that the probe solutionhas been applied to the right position on the carrier. Therefore, thenext liquid-ejecting operation has to be conducted before the probesolution applied to the carrier in the last liquid-ejecting operationdries and after visually confirming that the probe solution has beenapplied to the right position to make the manufacture of such a probecarrier disadvantageous. Furthermore, this technique is accompanied byadditional problems including that the operation of visually aligningthe carrier and the ink-jet head is time consuming and the number ofpoints to be used for observing the alignment of the carrier and theink-jet head is limited to make the alignment inaccurate. Moreover, ifthe probe carrier is turned upside down relative to the ink-jet headduring the operation of manufacturing the probe carrier, it cannot bechecked, if the carrier is transparent, simply by observing the carrier.

Japanese Patent Application No. 11-099000 discloses a method of fixingprobes at intended respective positions by forming a division wall (alsoreferred to as “black matrix”), or a light-shielding layer, on thecarrier typically by means of photolithography in order to enhance thecontrast produced by fluorescent light in the detection step. However,as a result of intensive research efforts, the inventors of the presentinvention found that, unless the ink-jet head is not accurately alignedrelative to the probe carrier, the probe solutions of adjacently locatedapertures (wells) with a division wall interposed therebetween canbecome mixed with each other (to produce a mixed solution) so that itmay no longer be possible to apply the proper probes to the intendedposition on the probe carrier. Then, the probe carrier will not functionproperly. Additionally, if the positions of ejection points aredisplaced, the probe solution in each well can be spread unevenly toexpose the surface of the probe carrier over a large area. Then, therearise problems such as unreliable detection, difficulty ofquantification, appearance of bright blank areas (also referred tosimply as “blank areas”) and an increase of unspecified bonds. “Blankareas” are produced when the ejected probe solutions do notsatisfactorily wet the probe carrier nor spread in the respectiveregions enclosed by a division wall. Then, probes will not be formeduniformly on the probe carrier as a result of applying probe solutionsto the latter. When probes that are not uniformly formed are subjectedto hybridization with respective specimens such as DNAs, the latter maynot only become mated with the respective probes having a specific basesequence but also adhere to the substrate (e.g., glass) that is exposedin the blank areas to consequently reduce the contrast produced byfluorescent light during the operation of observation. FIGS. 3A and 3Bof the accompanying drawings schematically illustrate this problem. InFIGS. 3A and 3B, reference symbols 31, 32, 33 and 34 respectively denotea transparent substrate, a division wall, a probe solution and a blankarea. FIG. 3B is a cross sectional view taken along line 3B-3B in FIG.3A.

Referring to FIGS. 3A and 3B, a blank area appears when a probe solution33 does not wet corners showing a complex profile and/or a narrowlyopened region nor spread well or when the probe solution 33 is appliedthinly near the surrounding division wall 32. While the phenomenon ofappearance of blank areas may be reduced when probe solutions areapplied at an enhanced rate, it will not be totally eliminated.

Photoresist is typically used for forming a division wall 32. Therefore,various ingredients of photoresist can adhere to and remain in theinside of the openings of the division wall 32. The adherent residuescan prevent probe solutions from wetting the probe carrier andspreading. Therefore, improvements are needed to obtain highly reliablefine probe carriers that can be manufactured with a high yield.

Furthermore, it is a problem to be solved urgently that the timerequired for aligning the ink-jet head and the probe carrier needs to bereduced because the operation of adjusting their relative positions andthe use of a detector for detecting the overall profile of the blackmatrix for the purpose of alignment are time consuming and hence raisethe manufacturing cost per chip.

SUMMARY OF THE INVENTION

In view of the above identified recent technological problems, it istherefore an object of the present invention to provide a probe fixingcarrier that allows the operation of aligning the ejection head and thecarrier to be conducted accurately and quickly when ejecting probesolutions onto the probe fixing carrier by means of an ink-jet systemand also the operation of locating a target substance accurately andquickly when detecting and quantifying the latter.

Another object of the present invention is to provide a probe carrierthat can effectively prevent the probe solutions being applied torespective probe fixing regions that are located adjacently and enclosedby a division wall from being mixed with each other and allows the probesolutions to sufficiently spread in the respective regions so as toprevent blank areas from being produced therein so that reliable probecarriers may be manufactured at a high yield.

In a first aspect of the invention, the above objects and other objectsof the invention are achieved by providing a method of manufacturing aprobe carrier carrying probes of a plurality of species fixed atrespective specific different positions on the carrier and adapted to bespecifically bonded to a target substance, said method comprising:

-   -   a step of forming one or more than one indexes on said carrier        at a position out of said specific positions; and    -   a step of applying solutions respectively containing said probes        to said respective specific positions by referring to said        indexes.

In a second aspect of the invention, there is provided a probe carriercarrying probes of a plurality of species fixed at respective specificdifferent positions on the carrier and adapted to be specifically bondedto a target substance, said probe carrier comprising:

-   -   one or more than one indexes formed on said carrier at positions        out of said specific positions.

In a third aspect of the invention, there is provided a probe fixingcarrier for carrying probes of a plurality of species fixed atrespective specific different positions on the carrier and adapted to bespecifically bonded to a target substance, said probe fixing carriercomprising:

-   -   one or more than one indexes formed on said carrier at positions        out of said specific positions.

In a fourth aspect of the invention, there is provided a method oflocating the position of the probe specifically bonded to a targetsubstance out of a number of probes of a plurality of species by causingsaid probes of a plurality of species carried on the carrier to contactthe target substance, said method comprising:

-   -   a step of locating the position of the probe specifically bonded        to the target substance by referring to indexes formed at        positions out of the specific positions carrying said probes of        a plurality of species.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1, 1A and 1B are schematic views of an embodiment of probe carrieror probe fixing carrier according to the invention and having indexesformed thereon.

FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G and 2H are schematic views of a probecarrier, illustrating different manufacturing steps of the method ofmanufacturing a probe carrier according to the invention.

FIGS. 3A and 3B are schematic illustrations of a blank area that can beproduced by a method of manufacturing a probe carrier, using an ink-jetsystem.

FIG. 4 is a schematic cross sectional view of an embodiment of probecarrier according to the invention.

FIGS. 5A and 5B are schematic cross sectional views of anotherembodiment of probe carrier according to the invention.

FIG. 6 is a schematic cross sectional view of still another embodimentof probe carrier according to the invention.

FIG. 7 is a schematic illustration of a plasma generator that can beused for a method of manufacturing a probe carrier according to theinvention.

FIG. 8 is a schematic illustration of another plasma generator that canalso be used for a method of manufacturing a probe carrier according tothe invention.

FIG. 9 is a schematic block diagram of the controller of a probe carriermanufacturing apparatus that can be used for the purpose of the presentinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Now, the present invention will be described in greater detail byreferring to the accompanying drawings that illustrate preferredembodiments of the invention.

FIGS. 1, 1A and 1B are schematic views of an embodiment of probe carrieror probe fixing carrier according to the invention and having indexesformed thereon. Referring to FIGS. 1, 1A and 1B, there are shown indexes11, a black matrix 12, openings (wells) 13, a chip 14 and a peripheralarea 15 of the chip 14. In the case of FIGS. 1, 1A and 1B, the indexes11 are surrounded by the black matrix 12 to produce recessed openings.Note that FIGS. 1, 1A and 1B only show an example of index arrangement.Firstly, indexes to be used for manufacturing a probe carrier accordingto the invention will be described in greater detail below. The term of“probe” as used herein refers to a single stranded nucleic acid having abase sequence complementary relative to all or part of the base sequenceof a target nucleic acid and adapted to detect the target nucleic acidas a result of being specifically hybridized with the base sequence ofthe target nucleic acid.

The indexes that are utilized when manufacturing a probe carrieraccording to the invention are typically used for the purpose ofaligning and positionally regulating the carrier and the probeapplication means when fixing the probe. In the case of the illustratedembodiment, probe solutions containing probes are ejected from anejection head onto a carrier and the positions on the probe carrierwhere the solutions are applied can be accurately located by using theindexes as alignment marks when fixing the probes on the probe carrierby means of an ink-jet system. With this arrangement, the positionswhere ejected liquid is applied to the probe carrier can be accuratelylocated and the alignment of the ejection head and the carrier can beregulated quickly, although the present invention is by no means limitedto the above described arrangement. Any method that involves relativepositional regulation of the carrier and the probe application means atthe time of fixation may be used for the purpose of the presentinvention. Additionally, the indexes may also be used as referencepoints for obtaining alignment information on the scanned image oraddress information on the positions of the plurality of probes at thetime of the detecting operation. The present invention providesremarkable advantages for achieving such combined objectives. Forexample, the indexes may be used firstly as alignment marks for aligningthe carrier and the probe application means at the time of fixing theprobes on the carrier and subsequently as alignment marks for detectingprobes and obtaining address information on the probes. In short, theindexes can be used for all the operations related to the probe carrier.

The indexes may be formed to show a projecting or recessed profilerelative to the carrier. They may alternatively be formed in thecarrier. For example, projections having a recessed profile may beformed on the carrier. Such projections may be formed by using resin andmade to show a desired profile by photolithography. If the carrier ismade of glass, indexes may be formed directly in the carrier by lasercutting or diamond cutting, although the indexes are preferably formedby using resin from the viewpoint of ease of detection. Preferably, theindexes are formed as openings of a division wall that is formed on thecarrier.

In the case of a chip where a plurality of probes are arrangedtwo-dimensionally and fixed, fixing regions are preferably formed bysuch a division wall to show a matrix as illustrated in FIG. 1. Then,indexes are formed in a non-fixing region having a profile differentfrom the members of the division wall so that the operation of relativepositional regulation of the carrier and the probe application means canbe conducted accurately and the liquid ejected onto the carrier can beprevented from unnecessarily swelling from the right regions by thedivision wall if the ejected liquid is inadvertently displaced by gasflows. As a result, the ejected solutions will be accurately andreliably applied to the respective openings. With this arrangement, itis only necessary to detect the indexes by appropriate detection meansso that the operation of relative positional regulation of the carrierand the probe application means can be carried out in a short period oftime to reduce the time necessary for manufacturing the chip if comparedwith the conventional alignment method having an initial step ofdetecting positional information on the entire chip (e.g., by visualdetection or by means of a CCD).

The division wall is preferably made of a resin or metal compositionhaving a light-shielding effect. A method of producing indexes havingsuch a division wall will be described in greater detail hereinafter.

Each probe array is provided with at least an index. A DNA chip isformed by combining a number of probe arrays. From the viewpoint ofaligning the ink-jet head and the carrier at the time of ejecting probesolutions by means of an ink-jet system and detecting the position ofthe target substance accurately and quickly, it is desired that aplurality of indexes are proved on each chip. Accurate two-dimensionalpositional information can be obtained by scanning two or more than twoindexes. Additionally, if the ejection head is inclined inaccuratelyrelative to the carrier, the inclination of the ejection head can beadjusted by referring to the two or more than two indexes. The pluralityof indexes do not need to show a same and identical profile. Indexeshaving different shapes may be used for the purpose of the invention.

Indexes may be formed for each probe array at any positions so long asthey are located outside the probe fixing regions (on the carrier wherethe respective probes are fixed) as shown in FIGS. 1, 1A and 1B. Theymay be arranged in a peripheral area of the chip as in the case of FIGS.1, 1A and 1B, in areas located between adjacent probes or in a divisionwall separating openings (wells) if the division wall is formed on thecarrier. When indexes are formed in areas located between adjacentprobes, the peripheral area of the chip may also be used for probefixing regions so that more probes can be mounted on the chip to raisethe density of probe arrangement. If, on the other hand, indexes areformed in the peripheral area of the chip, they are preferably formed atpositions located as close as possible to the probe fixing regions fromthe viewpoint of accurate alignment.

While each index may have any form, it is preferably made to show aprofile that allows an accurate and quick alignment of the ejection headand the carrier for the operation of ejecting probe solutions and aneasy detection of the position of the target substance. Each index mayhave the form of a cross, a circle, a square, a rectangle, an isoscelestriangle, a butterfly or the like. The form of a cross provides anadvantage that it can be formed with ease in the chip manufacturingprocess and its center can be located easily. A cross-shaped indexpreferably has a transversally part that is 30 μm wide and 150 μm longand a vertical part that is also 30 μm wide and 150 μm long.

The dimensions of each index can be modified appropriately depending onthe required accuracy of alignment and positional detection. When adivision wall is arranged on the carrier, each index may typically bemade to have a size substantially same as that of each opening (well).If indexes are formed at the time of forming a division wall, they havea thickness same that of the latter. Then, the thickness is preferablyfound within a range between 0.5 μm and 100 μm.

An alignment mark that is used with the method of manufacturing a probecarrier according to the invention can be utilized for both aligning thecarrier and the probe application means at the time of preparing theprobe carrier and detecting the position of the target substance. Inother words, separate indexes do not need to be provided for the twooperations so that consequently the area required for a probe non-fixingregion can be reduced. Furthermore, the index can be used as mark fordiscriminating the front surface and the back surface of the carrier.

For the purpose of fixing probes, it is not necessary that all the wellsare provided with respective probes. In other words, the number, thetypes and the positions of probes may be modified appropriately.Preferably, information on the positions where probes are fixed can beobtained also by referring to the indexes. For instance, the profilesand the number of the indexes may be modified according to the numberand the types of probes to be fixed and information on the positions ofthe probes can be obtained at the time of the target substance detectingoperation.

When a division wall is provided on the carrier and the material usedfor forming the division wall has a light-shielding effect, the divisionwall provides a light-shielding effect during the detecting operationinvolving the use of fluorescent light so that the indexes functionsatisfactorily during the operation of observation provided thatopenings of division wall are used as indexes. The alignment operationcan be conducted satisfactorily regardless of the environment of theoperation when a material that may be a fluorescent substance andoperates advantageously for the operation of observation is provided onthe openings that are formed by members having a light-shielding effect.

Now, embodiments of probe fixing carrier and probe carrier carryingprobes and a method of manufacturing such a carrier according to theinvention will be described by referring to the accompanying drawingsparticularly in terms of a case where the probe non-fixing regionoccupies a division wall.

FIGS. 2A through 2H are schematic views of a probe carrier, illustratingdifferent manufacturing steps of the method of manufacturing a probecarrier according to the invention. Now, the steps will be describedbelow. In FIGS. 2A through 2H, there are shown an index 11, a divisionwall 12 having the index 11, wells 13, a carrier 21, a division walllayer 22 preferably made of a resin or metal composition, a photoresistmask 23, a probe solution ejection head 24 and probe solutions 25. FIG.2A is a schematic plan view of the embodiment, where the index 11 iscross-shaped and surrounded by the division wall 12 to form a recessedopening. While the center of the opening is made to agree with thecenter of the cross for the purpose of convenience, the presentinvention is by no means limited to such an arrangement.

Step (a): a Carrier for Probe Arrays.

A probe fixing carrier 21 is prepared (see FIG. 2B). The expression of a“carrier” for probe arrays as used herein refers to one for carryingprobes. Hence, any carrier that can carry probes and does not obstructthe operation of detecting a target substance can be used for thepurpose of the invention. Materials that can be used for a carrier forprobe arrays include glass substrates, silicon substrates, metalsubstrates and resin substrates such as acryl resin substrates as wellas hollow objects and tubular carriers. A carrier for probe arrays mayor may not be surface-treated for hydrophilicity. An opticallytransparent substrate or, in some cases, an optically black substratemay preferably used when a reaction is optically detected. Carriers thatcan preferably be used for the purpose of the invention include glasssubstrates such as synthetic quartz substrates and fused quartzsubstrates, silicon wafers and resin substrates such as acrylsubstrates, polycarbonate substrates, polystyrene substrates and vinylchloride substrates, to which black pigment or dye may or may not beadded. Black pigments that can be used for the purpose of the inventioninclude carbon black and organic black pigments. Preferably, the carriersurface is treated in advance so as to make it have a structure adaptedto react with various organic functional groups to be introduced toprobes and form a covalent bond and also receive one or more than oneorganic functional groups. Examples of preferable combinations of afunctional group introduced to a terminal of the probe and a functionalgroup to be introduced to the carrier surface so as to be bonded withthe former functional group include a combination of thiol (—SH)(nucleic acid probe terminal) and a maleimide group (carrier surface)and a combination of an amino group (nucleic acid probe terminal) and anepoxy group (carrier surface).

The surface of the carrier 21 may be subjected to a surface treatment,which may be a plasma treatment, a UV treatment, a coupling treatment ora treatment with albumin.

Step (b): Preparation of a Resin Composition Layer

Then, a division wall layer 22 is formed on the carrier 21 in order toproduce a division wall 12 for forming wells 13 and indexes 11 that areopenings (see FIG. 2B). When the division wall 12 is a blacklight-shielding layer, it may also be referred to as black matrix.

The division wall 12 is preferably a light-shielding layer that blockslight between any adjacent probe fixing regions. Then, the division wall12 may be realized in the form of a black matrix or black stripes. Asthe division wall 12 is made to operate as a light-shielding layer, thedetection accuracy (SN ratio) can be remarkably improved particularlywhen the hybridization of a probe and a target substance on the carrieris optically detected (by detecting fluorescent light).

A metal selected from chromium, aluminum and gold may be used for thedivision wall layer 22. The use of black chromium is preferable from theviewpoint of reliability particularly when it is combinedly used with atransparent substrate for an optically detecting operation because it ishighly light-shielding. However, it should be noted that metal is moreoften than not hydrophilic and the division wall layer 22 is generallymade to have a film thickness of several thousands angstroms from theviewpoint of uniform film thickness when it is formed by evaporation orsome other similar technique. These characteristics of a metal divisionwall layer 22 need to be taken into consideration when it is used.

A material that is hydrophobic relative to the substrate isadvantageously be used for forming the division wall layer 22. Examplesof materials that can be used for the division wall layer 22 includeepoxy type resins, acryl type resins and polyimide (or polyimideamide)type resins, urethane type resins, polyester type resins and polyvinyltype resins, which may or may not be photosensitive. The division walllayer 22 preferably is resistant against hot temperature equal to orhigher than 250° C. The use of epoxy type resins, acryl type resins orpolyimide type resins is preferable from the viewpoint of thermalresistance.

The division wall 12 can be made to be an advantageous light-shieldinglayer by using a resin composition layer 22 that is made of a blackresin composition where a light-shielding agent is dispersed. Carbonblack is preferably used as light-shielding agent from the viewpoint ofproviding the division wall 12 with an enhanced level of waterrepellency and an appropriate degree of surface coarseness. Carbon blackmaterials that can be used for the purpose of the present inventioninclude those produced by way of a contact process such as channelblack, roller black and disk black, those produced by way of a furnacedprocess such as gas furnaced black and oil furnaced black and thoseproduced by way of a thermal process such as thermal black and acetyleneblack, of which the use of channel black, gas furnaced black or oilfurnaced black is particularly favorable. Black organic pigment may beused alternatively. It is also possible to use commercially availableblack resist.

The division wall layer 22 can be formed by way of an appropriateprocess selected from spin coating, roll coating, bar coating, spraycoating, dip coating and printing.

If no division wall 12 is formed on the carrier, the above division wallforming process will be omitted as a matter of course.

Step (c): Preparation of Division Wall and Alignment

The division wall 12 is formed simultaneously for both the indexes 11and the wells 13 by photolithography (see FIG. 2C).

With a method of forming the pattern of the division wall 12 and that ofthe indexes 11, a photoresist mask 23 having the pattern of the wells 13and that of the indexes 11 is laid on the resin coating the surface ofthe carrier 21 in the above step (b), and the resin layer is selectivelyexposed to light and developed for the purpose of patterning in order toproduce a division wall 12 having a plurality of wells 13 that are probefixing regions and indexes showing a recessed profile. The profiles, thepositions and the numbers of indexes 11 are described above in detail.Thus, the indexes 11 are produced by using a photoresist mask showing apattern corresponding to that of the indexes. If a photosensitive resinis used, the resin itself may be used as photo mask and subjected to apatterning operation, using a photolithography process. Photosensitiveresin materials that can be used for the purpose of the inventioninclude UV resist, DEEP-UV resist and UV setting resins. The divisionwall of the black matrix is preferably made to become water repellenttypically by means of a dry etching operation that is conducted afterthe patterning operation. If a photosensitive material is used for theresin composition layer 22, the water repellency of the division wall 12can be improved by baking the pattern obtained by patterning the resinlayer 22. A major advantage of making the division wall 12 show waterrepellency is that probe solutions can be applied smoothly to theintended respective wells if the probe solutions hit the respectiveopenings with a slight degree of positional displacement. Note that theopenings 13 are usually formed as wells.

The profile of the division wall openings 13 that operate as probefixing regions can be appropriately defined by taking the easiness offorming and handling and the operability at the time of detection of thetarget substance into consideration. While the profile is notparticularly limited and may be polygonal or elliptic, it is preferablethat the division wall openings 13 show a simple profile such as square,rectangular or circular. The arrangement of the openings on the carriermay be appropriately modified if desired.

The surface area of the probe solution that is applied to a singleopening (well) 13 is generally between 0.01 μm² (e.g., 0.1 μm×0.1 μm)and 40,000 μm² (e.g., 200 μm×200 μm), although it is defined as afunction of the size of the array itself and the density of the arraymatrix. Therefore, the size and the capacity of each opening (well) towhich a probe solution is applied can be determined on the basis of thesurface area of the probe solution. The depth of each opening (well) ispreferably between 0.5 μm and 100 μm, although it may vary depending onthe process selected for producing the openings (wells). The volume(capacity) of each opening is defined by the area and the depth thereof.For example, a method as described in Japanese Patent Application No.11-099000 may be used without modification for producing such openings(wells).

The division wall 52 may show a trapezoidal cross section as shown inFIG. 5A. FIG. 5B is an enlarged cross sectional view of a part of thedivision wall of FIG. 5A. Alternatively, the division wall may show aninverted trapezoidal cross section as shown in FIG. 6.

Referring to the cross sectional view of a division wall of FIG. 5B, A1and A2 indicate the part of the division wall located at 80 to 100% ofthe height of the top of the division wall as viewed from the bottom ofthe cross section of the division wall and B1 and B2 indicate the partof the division wall that directly contacts the carrier as shown in thecross section of the division wall. In FIG. 5B, L1 is the distancebetween A1 and A2 and L2 is the distance between B1 and B2 while Tindicates the height of the top of the division wall and T′ indicatesthe height of A1 and A2 showing the part of the division wall located at80 to 100% of the height of the top of the division wall. The spanbetween A1 and B1 and the span between A2 and B2 are referred to aslateral surfaces of the division wall herein.

If the average value of the length L1 that is parallel to the substrateand represents the part of the division wall located at the height T′that is 80 to 100% of the height T of the top of the division wall asmeasured from the bottom to the top of the cross section of the divisionwall 52 is ‘a’ and the length L2 of the bottom of the cross section ofthe division wall 52 is ‘b’, it is preferable that the relationship of aand b satisfies the requirement of formula 1.3≧a/b≧0.7-in the crosssection of the division wall 52. If the ratio of a/b is smaller than0.7, uneven probes are formed by probe solutions. Such probes areundesirable from the viewpoint of uniformity of probes and can give riseto a problem of blank areas. The ratio of a/b can be made greater than 1as shown in FIG. 6. Then, the cross section of the division wall 62 isinversely trapezoidal. Such an arrangement is advantageous because eachprobe solution is partly hidden by the corresponding umbrella of thedivision wall 62 to reduce the “blank area”, if any, at the time ofdetection. Note, however, the entire structure will become unstable whenthe ratio of a/b is excessively large. It is preferable that a/b is notgreater than 1.3.

If no division wall 12 is formed on the carrier, this step is omittedand desired indexes are simply formed. As described above, projectionshaving a recessed profile may be formed on the carrier by using a resinmaterial and a photolithography process. Alternatively, if the carrier21 is a glass substrate, indexes having a recessed profile may bedirectly formed in the substrate by laser cutting or by means of adiamond cutter.

Step (d): Dry Etching Treatment

The carrier 21 on which a division wall 12 is formed is preferablysubjected to a dry etching treatment (see FIG. 2D). More specifically,gas containing at least oxygen, argon or helium is introduced for areduced pressure plasma treatment process in which the carrier 1 isirradiated with plasma in a reduced pressure environment or anatmospheric pressure plasma treatment process in which the carrier 1 isirradiated with plasma in an atmospheric pressure environment.

As a result of the dry etching treatment, polutant that comes to adhereto the surface of the carrier 21 in the step of forming the divisionwall 12 is removed and the surface is cleaned to show affinity for theprobe solutions 25 applied to it in a subsequent step so that the probesolutions 25 may be effectively dispersed in the respective openings 13after a subsequent water treatment step. Further more, the surface ofthe division wall 12 is made coarse to increase its water repellency asa result of the dry etching treatment.

Step (e): Plasma Treatment

The carrier 21 that is subjected to a dry etching treatment, ifnecessary, is subsequently subjected to a plasma treatment where thecarrier 21 is irradiated with plasma in a gas atmosphere containing atleast fluorine atoms (see FIG. 2E). As a result of the plasma treatment,fluorine or a fluorine compound in the introduced gas penetrates thesurface layer of the division wall 12 to increase the water repellencyof the surface of the division wall 12.

A particularly high degree of water repellency is realized when thedivision wall 12 is formed by using a resin composition containingcarbon black. The reason for this may be that carbon black comes to beexposed to the surface of the division wall 12 as a result of the dryetching treatment of step (d) and chemically bonded with fluorine or thefluorine compound as a result of the plasma treatment of this step.Therefore, it is desirable that the division wall 12 is made to containcarbon black for the purpose of the present invention.

One or more than one halogen gases selected from CF₄, CHF₃, C₂F₆, SF₆,C₃F₈ and C₅F₈ are preferably used as gas containing at least fluorineatoms and introduced in this step. Particularly, the use of C₅F₈(octafluorocyclopentene) is advantageous because it does not destroyozone at all and, at the same time, shows a short life of 0.98 years inthe atmosphere, which is very short if compared with other gases (CF₄:50,000 years, C₄F₈: 3,200 years). Thus, it shows an earth warmingcoefficient of 90 (accumulated value for 100 years as determined on thebasis of CO₂=2), which is also very small if compared with other gases(CF₄: 6,500, C₄F₈: 8,700). Therefore, the use of C₅F₈ is veryadvantageous from the viewpoint of protection of the ozone layer and theenvironment of this planet. The use of this gas is highly recommendedfor the purpose of the present invention.

If necessary, oxygen, argon and/or helium may be added to the gasintroduced in this step. The degree of water repellency that is obtainedin this step can be controlled by using a mixture gas containing one ormore than one halogen gases selected from CF₄, CHF₃, C₂F₆, SF₆, C₃F₈ andC₅F₈ and O₂. However, the oxidizing reaction of O₂ becomes dominant toobstruct the effort for enhancing the water repellency and the resin canbe remarkably damaged if the mixing ratio of O₂ exceeds 30%. Therefore,the mixing ratio of O₂ needs to be less than 30% if a mixture gas isused in this step.

Low frequency discharge, high frequency discharge, microwave dischargeor some other appropriate technique may be used for generating plasmafor the purpose of this step and the preceding step of dry etchingprocess. The pressure, the gas flow rate, the discharge frequency, theprocessing time and other conditions of the plasma irradiation may beselected appropriately.

FIGS. 7 and 8 schematically illustrate plasma generators that can beused for the dry etching process and the plasma treatment of the presentinvention. In each of FIGS. 7 and 8, there are shown an upper electrode71, a lower electrode 72, a carrier 73 to be treated and a highfrequency power source 74. In each of the illustrated plasma generators,a high frequency voltage is applied to the two electrodes that areplate-shaped and arranged in parallel with each other to generateplasma. More specifically, FIG. 7 shows a plasma generator of thecathode coupling type, whereas FIG. 8 shows a plasma generator of theanode coupling type. With either system, the water repellency and thecoarseness of the surface of the division wall 12 and the affinity forprobe solutions of the surface of the carrier 21 can be made to show adesired degree by controlling the pressure, the gas flow rate, thedischarge frequency, the processing time and other conditions of theplasma irradiation.

With the plasma generator of the cathode coupling type of FIG. 7, it ispossible to reduce the duration of the dry etching treatment and henceis advantageous in this sense. On the other hand, the plasma generatorof the anode coupling type of FIG. 8 is advantageous in that it does notunnecessarily damage the carrier 21. Therefore, either of the plasmagenerators may appropriately be selected for the dry etching treatmentand the plasma treatment depending on the material of the carrier 21 andthat of the division wall 12.

As a result of this step, the amount of the fluorine compound existingon the surface of the carrier 21 that is exposed in the openings 13 ofthe division wall 12 is reduced to less than a half of the originalamount and the surface coarseness of the exposed surface issignificantly raised if compared with the surface before this step.

Note that the effect of spreading probe solutions of the carrier isreduced if the carrier 21 is brought into contact with water andsubsequently heated and dried at temperature exceeding 100° C. in thisstep. Therefore, the drying operation should be conducted at temperaturelower than 100° C.

Step (f): Water Treatment

The surface of the carrier 21 that is subjected to a plasma treatment isthen brought into contact with water for a water treatment for thepurpose of improving the effect of spreading probe solutions of thecarrier 21 in the openings 13 of the division wall 12 (see FIG. 2F). Asa result of this treatment, a probe solution can be made to sufficientlyspread within an opening 13 if the amount of the applied probe solutionis very small.

Preferably, pure water is used for the water treatment of this step. Anymethod may be used for bringing the carrier 21 into contact with waterso long as it can perfectly bring them into mutual contact. Methods thatcan be used for this step include immersion into water and washing withwater. However, if the division wall 12 shows a complex pattern on thecarrier 21, the carrier 21 is preferably dipped into water andirradiated with ultrasonic waves simultaneously or washed with finewater drops under high pressure so that boundaries of the division wall12 and the openings 13 and fine and delicate parts such as corners ofthe openings 13 may sufficiently contact with water.

While the temperature of water that is brought into contact with thecarrier 21 is preferably high from the viewpoint of effectivelyimproving the surface condition of the openings 13, it is advantageouslybetween 20 and 60° C. from the viewpoint of economic efficiency ofheating water.

Now, the water repellency and the hydrophilicity of the division walland the probe fixing regions (openings) after the water treatment stepwill be discussed below. According to the invention, the division wallis made of a resin composition containing carbon black and then it ispossible to provide a high contact angle of 120° or more for the waterrepellency of the surface of the division wall after the steps of plasmatreatment and water treatment. According to the invention, the contactangle of water on the surface of the division wall 12 is more than 120°,preferably more than 135°, more preferably more than 150°, mostpreferably 180°, for the water repellency of the surface of the divisionwall 12 relative to pure water after the water treatment step. A mixedsolution is liable to be produced so that probe solutions can not beapplied to a large extent if the contact angle is less than 120°.Particularly, it will be difficult to manufacture a probe carriershowing a high detection sensitivity if the contact angle is less than120°. With any comparable conventional methods, it is difficult to makethe contact angle higher than 120° for the water repellency of thesurface of the division wall and the contact angle is slightly less than110° even if the probe carrier is made of PTFE (polytetrafluoroethylene)that is a highly water repellent material. To the contrary, according tothe invention, probe solutions can be effectively prevented from beingmixed with each other as the contact angle is made greater than 120° forthe water repellency of the surface of the division wall.

As for the hydrophilicity of the openings 13 of the carrier 21, thecontact angle is desirably not greater than 30° when measured by usingpure water. As the contact angle is made less than 30° relative to probesolutions, the polutant that is made to adhere to the surface of thecarrier 21 exposed in the openings 13 in the dry etching treatment ofstep (d) is removed to improve the ink spreading effect that is endowedin the subsequent water treatment step. Particularly, the contact angleis preferably not greater than 20° when measured by using pure water.

With regard to the water repellency of the division wall and thehydrophilicity of the probe fixing regions in the openings 13 of a probefixing carrier 21 according to the invention and prepared in a manner asdescribed above, the contact angle relative to each probe solution mayvary depending on the solvent contained in the probe solution. Anaqueous solution containing glycerol by 7.5 wt %, urea by 7.5 wt %,thiodiglycol by 7.5 wt % and acetylene alcohol expressed by generalformula (VII):

by 1 wt % is preferably used as the solvent of each probe solution. (Inthe above formula (IV), each of R₁, R₂, R₃ and R₄ represents a straightchain or branched alkyl group having 1 to 4 carbon atoms, while each ofm and n represents 0 or a positive integer and m+n≦30.) When an abovedefined probe solution is used, the contact angle of the division wallrelative to the probe solution can be made to be greater than 90° in aprobe fixing carrier manufactured according to the invention thatcomprises a division wall 12 made of a resin composition containingcarbon black and is subjected to a dry etching treatment and asubsequent plasma treatment. With such a contact angle, the surface ofthe probe fixing carrier shows a sufficiently high degree of waterrepellency relative to the probe solution so that occurrence of a mixedsolution and appearance of blank areas can be effectively prevented. Thecontact angle is preferably greater than 100°, more preferably greaterthan 115°, most preferably greater than 130°. The contact angle of eachof the openings 13 relative to the corresponding probe solution ispreferably less than 30°. With such a contact angle, it is possible toachieve a sufficient improvement for the liquid spreading effect as aresult of the water treatment. More preferably, the contact angle isless than 20°.Step (g): Application and Fixation of Probe Solutions onto the Carrier

Probe solutions are applied to the carrier and fixed there in a manneras described below. After regulating (aligning) the position of theliquid ejection head 24 relative to the carrier, probe solutions 25 areapplied to the respective wells 13 in the division wall 12 by means ofan ink-jet type apparatus (see FIGS. 2G and 2H). Now, an embodiment thatis suitable for application and fixation of probe solutions onto thecarrier will be described below in detail.

(Application Alignment)

With application alignment, the indexes prepared in the above describedmanner are shot by an imaging means such as a CCD camera adapted to usea laser as light source and the obtained image is processed andrecognized by an image processing device to detect the positionalcoordinates of the indexes. Then, the XYθ stage of the liquid ejectiondevice is positionally regulated on the basis of the detectedcoordinates and the head and the carrier are aligned. If it is desirableto reduce the dimensions of the indexes particularly in terms of theline width thereof, a CCD camera comprising an area sensor having alarge number of pixels and/or provided with a plurality of area sensorsmay preferably be used for the purpose of the alignment.

(Liquid Ejection Device)

A probe carrier manufacturing apparatus to be used with a method ofmanufacturing a probe carrier according to the invention comprises aliquid ejection means for ejecting probe solutions by way of an ink-jetsystem and is adapted to apply solutions containing respective probes tospecific respective positions on the probe fixing carrier having indexesin a probe non-fixing region thereof by referring to the positions ofthe indexes.

More specifically, while a probe carrier manufacturing apparatus to beused with a method of manufacturing a probe carrier according to theinvention is provided with a liquid ejection means for ejecting probesolutions by way of an ink-jet system, the liquid ejection mechanism ofthe apparatus by turn comprises at least as many liquid ejectingsections as the number of probe solutions of a plurality of species.Each of the liquid ejecting sections has a liquid containing section forcontaining a probe solution, an ejection port provided for the liquidcontaining section, a liquid path for making the liquid containingsection communicate to the corresponding ejection port and an energygenerating means arranged at the ejection port so as to make it possiblefor the latter to eject the probe solution independently from theremaining probe solutions. The liquid ejection mechanism furthercomprises a means for detecting the positions of the indexes. The liquidejection mechanism is preferably so designed that, after aligning thehead and the carrier at desired respective positions by referring to theindexes, the probe solutions of a plurality of species are ejectedindependently from the liquid ejection means onto the carrier, whilemoving the liquid ejection means relative to the carrier that hasrecesses corresponding to the respective probe receiving positions, onthe basis of the information on the positional arrangement of theprobes. On the other hand, the arrangement for detecting the positionsof the indexes is formed by an imaging means such as a CCD camera forobtaining an image of the surface of the probe array carrier, an imageprocessing means and an XYθ stage for regulating the relative positionsof the liquid ejection means and the probe carrier.

FIG. 9 is a schematic block diagram of the controller of a probe carriermanufacturing apparatus 101 that can be used for the purpose of thepresent invention. Referring to FIG. 9, there are shown an interface 95for exchanging data with teaching pendant 92, a CPU 96 for controllingthe probe carrier manufacturing apparatus 101 and processing theobtained image, a ROM 97 storing control programs necessary foroperating the CPU 96, a RAM 98 for storing information on abnormalconditions, an ejection control section 99 for controlling the ejectionof probe solutions, a stage control section 100 for control theoperation of the XY stage (not shown) of the probe carrier manufacturingapparatus 101 and the probe carrier manufacturing apparatus 101 that isconnected to the controller 91 and operate according to the instructionsgiven by the controller 91.

(Probe Solution Ejection Head)

The liquid ejection head that is used for a method of manufacturing aprobe carrier according to the invention is adapted to eject probesolutions by utilizing thermal energy. Therefore, preferably it isprovided with a thermal energy generator for generating thermal energyto be applied to probe solutions. The ink-jet system that can be usedfor manufacturing a probe carrier may be a thermal jet system (bubblejet system), with which liquid is ejected by way of generating a bubble,utilizing thermal energy generated from electro-thermal converters suchas heaters or lasers, or a piezo jet system adapted to eject liquid byway of the deformation of a piezo-element produced by applying a voltageto the element. Either system may be used for the purpose of the presentinvention. Of the two systems, the head to be used with the thermal jetsystem has a structure relatively simple if compared with the head to beused with the piezo jet system and hence can suitably be downsized andprovided with a multi-nozzle. Additionally, the time required for probesolutions to be bonded to the carrier is relatively short so thatproduction of a secondary structure of DNA can be avoided by the heatinvolved in the thermal jet system and the efficiency of the subsequenthybridization can be improved. For these reasons, the thermal jet systemis advantageously used as the ink-jet system for the purpose of thepresent invention.

Typically, it is advantageous for the purpose of the invention toutilize the basic principles in the field of ink recording that aredisclosed in U.S. Pat. Nos. 4,723,129 and 4,740,796. The disclosedprinciples may be applied to both the so-called on-demand type and thecontinuous type. However, the use of the on-demand type may beadvantageous for the purpose of the invention because thermal energy canbe generated in the electro-thermal converters that are arranged incorrespondence to the liquid (ink)-holding sheets or the liquid paths byapplying at least a drive signal for causing a rapid temperature rise toa level exceeding nucleus boiling in correspondence to recordinginformation in order to give rise to film boiling to the thermal actionplane of the recording head (liquid ejection head) and consequently abubble can be formed in the liquid (ink) to show one-to-onecorrespondence relative to the drive signal. Liquid (ink) is ejected byway of an ejection port as a result of growth and contraction of thebubble to produce at least a droplet. If the drive signal ispulse-shaped, the bubble can be made to grow and contract immediatelyand appropriately so that liquid (ink) can be ejected in a highlyresponsive manner to a great advantage of effective ejection of liquid(ink). A pulse-shaped drive signal as described in U.S. Pat. Nos.4,463,359 and 4,345,262 may suitably be used for the purpose of theinvention. The information recording effect may be further improved whenthe conditions for the temperature rising rate of the thermal actionplane as described in U.S. Pat. No. 4,313,124 are met.

As for the configuration of the ejection head, those realized on thebasis of U.S. Pat. Nos. 4,558,333 and 4,459,600 that disclose a thermalaction section arranged in a curved region in addition to the use of acombination of an ejection port, a liquid path and an electro-thermalconverter (a straight liquid flow path or a rectangular liquid flowpath) are within the scope of the present invention. Additionally, theadvantages of the present invention can be effectively exploited if theuse of a common slit for the ejection sections of a plurality ofelectro-thermal converters as disclosed in Japanese Patent ApplicationLaid-open No. 59-123670 and the arrangement of an open hole forabsorbing pressure waves of thermal energy in correspondence to theejection sections as disclosed in Japanese Patent Application Laid-openNo. 59-138461 are applied to the present invention. In short, regardlessof the configuration of the liquid ejection head, spotting of probes canbe accurately and efficiently realized according to the invention.

Furthermore, a full-line type liquid ejection head having a lengthcorresponding to the largest width of the carrier that can be spotted bya liquid ejection device can also advantageously be applied to thepresent invention. Such a liquid ejection head may be so configured asto adapt itself to the necessary length either by combining a pluralityof heads or by using an integral unitary head.

Furthermore, a serial type liquid ejection head rigidly secured to theapparatus main body, a replaceable tip type liquid ejection head that iselectrically connected to the apparatus main body as it is fitted to thelatter and adapted to apply probe solutions from the apparatus main bodyor a cartridge type liquid ejection head provided with an integralsolution reservoir can also advantageously be applied to the presentinvention.

Preferably, a liquid ejection apparatus comprising a liquid ejectionhead that is provided with an ejection restoring means and/or a spareauxiliary means is used for the purpose of the invention because such anapparatus can enhance the advantages of the invention. Specific examplesof such means include a cleaning means to be used for the liquidejection head, a pressurizing or suction means, a spare heating meansthat may be an electro-thermal converter or a heating element of adifferent type or a combination thereof and a spare ejection means thatis adapted to eject liquid in a form other than spotting.

Of the above listed devices, a device adapted to a film boiling systemis most advantageous for the above described solutions.

(Probe Solution)

Now, the compositions of probe solutions that are used when preparing aprobe carrier from a probe fixing carrier according to the invention andthe method of ejecting such probe solutions will be described in detailbelow.

According to the invention, the probes that are fixed to a probe fixingcarrier can be specifically bonded to a specific target substance.Additionally, the probes may contain oligonucleotides, polynucleotidesand/or other polymers that can be recognized by a specific target. Theterm of “probe” as used herein refers to individual molecules that canoperate as probe such as polynucleotide molecules or a group ofmolecules such as polynucleotide molecules that are fixed as distributedon a surface to show a same and identical arrangement and includesmolecules called ligands. The probe and the target can often beexchangeable and also can be bonded or made to become bonded to eachother as part of ligand-antiligant (which may also be referred to asreceptor) pairs. For the purpose of the invention, a probe and a targetcan contain one or more than one natural bases and/or analogs.

Examples of probe that is supported on a carrier include a substancehaving a bonding section that is to be bonded to the carrier in a partof the oligonucleotide thereof showing a base arrangement that can behybridized with a target nucleic acid, said bonding section to be bondedto the carrier being linked to the oligonucleotide by way of a linker.There are no limitations to the position of the bonding section to bebonded to the carrier in the oligonucleotide molecule so long as thedesired reaction of hybridization is not adversely affected.

While the probes carried by a probe carrier manufactured by a methodaccording to the invention may be appropriately selected depending onthe application thereof, it is preferable that one or more than onetypes of probes are selected from DNAs, RNAs, cDNAs (complementaryDNAs), PNAS, oligonucleotides, polynucleotides and other nucleic acids,oligopeptides, polypeptides, proteins, enzymes, substrates relative toenzymes, antibodies, epitopes relative to antibodies, antigens,hormones, hormone receptors, ligands, ligand receptors, oligosaccaridesand polysaccharides for the purpose of advantageously embodying themethod of the present invention.

For the purpose of the invention, a probe carrier refers to an objectrealized by fixing a plurality of probe species in independentrespective regions of the surface of the carrier (including the surfaceof the inner walls of hollow members or tubular carrier members)typically as dot-shaped spots, while a probe array referes to a probecarrier in which probes are arranged orderly at a predeterminedinterval.

On the other hand, each probe has a structure that makes it possible tobe bonded to the surface of the carrier and preferably be bonded ontothe carrier by way of the structure that makes it possible to be bondedto the surface thereof. Preferably, the structure of each probe thatmakes it possible to be bonded to the surface of the carrier is formedby means of a process of introducing an organic functional group such asa amino group, a mercapto group, a carboxyl group, a hydroxyl group, anacid halide (haloformyl group; —COX), a halide (—X), aziridine, amaleimide group, a succinimide group, isothiocyanate, a sulfonylchloride(—SO₂Cl) group, an aldehyde group (formyl group, —CHO), hydrazine oracetamide iodide. Whenever necessary, the surface of the carrier may betreated appropriately in a manner that depends on the structurenecessary for bonding the probes to the carrier.

When applying probe solutions containing probes onto a carrier by meansof an ink-jet system, it is preferable that the liquid compositions areprepared in such a way that the applied droplets of the solutions do notspread unnecessarily on the carrier but remain within a predeterminedarea. Additionally, the liquid compositions are such that they do notadversely affect the proper performance of the probes nor the propertyof reacting with the respective functional groups on the surface of thecarrier onto which the probes are introduced.

When an ink-jet system is used, it is preferable that the carrier isstored in a reaction vessel such as a wet vessel during the reaction inorder to prevent the applied droplets from evaporating and dryingbecause the droplets applied to the carrier surface are very small.Alternatively, the liquid ejected from the liquid ejection head may bemade to contain a humidstatic agent. Particularly, in the case of athermal jet system, the operation of ejecting liquid is accompanied by atemperature rise and hence the use of a humidstatic ingredient and asurface tension regulating ingredient is important. A solvent containingurea by 5 to 10 wt %, glycerol by 5 to 10 wt %, thiodiglycol by 5 to 10wt % and acetylene alcohol by 1 wt % is preferably used for the purposeof applying marker substances or probes to the surface of the carrier.

Considering the ink-jet performance of the ejected liquid and thestability of the probe in the liquid and at the time of thermal jet(bubble jet) ejection, it is preferable that each probe solutiontypically contains a nucleic acid probe of 2 mer to 5,000 mer,particularly 2 mer to 60 mer to a concentration of 0.05 to 500 μM,particularly 2 to 50 μM.

As for the physical properties of each probe solution as liquid, itpreferably shows a viscosity between 1 and 15 cps and a surface tensionof not less than 30 dyn/cm from the viewpoint of its performance whenejected from a thermal jet head (bubble jet head). The probe solutionwill accurately and advantageously hit the target position on thecarrier when the viscosity is between 1 and 5 cps and the surfacetension is between 30 and 50 dyn/cm.

However, the viscosity and the surface tension of the liquid and thebase length and the concentration of the nucleic acid probe may be outof the above defined respective ranges when a well-like structure isused so that the liquid applied onto the carrier by means of a liquidejection head is prevented from spreading on the carrier and becomemixed with the liquid of an adjacent spot.

(Ejection Mode)

The amount of probe solution that is ejected from a single nozzle at atime is selected as a function of the surface density of the probe spoton the probe carrier to which the probe solution is ejected. Morespecifically, the amount is selected as a function of the size and theshape of the dots of the probe array, taking the viscosity of the probesolution, the affinity between the probe solution and the carrier, thereactivity of the probe and the carrier and other elements intoconsideration. The amount of probe solution that is ejected from asingle nozzle at a time is between 0.1 and 100 pl (picoliter),preferably between 0.1 and 50 pl. While the amount that is ejected at atime is preferably minimized from the viewpoint of raising the densityof probes on the carrier, the probe solution will be prevented fromgetting to the carrier by the resistance of air if the amount that isejected at a time is too small. The amount that is ejected at a time ispreferably between 0.1 and 10 pl, more preferably between 0.1 and 5 pl.Therefore, the nozzle diameter and other factors need to be designed byconsidering the amount.

Each spot of nucleic acid probe preferably shows a diameter between 20and 100 μm and the spots are preferably made to be independent from eachother for the purpose of confining the density of nucleic acid probeswithin the above range (and arranging more than 10,000 spots per inchwith an upper limit of about 1×10⁶ spots). The spots are defined as afunction of the characteristics of the liquid ejected from each bubblejet head and those of the surface of the carrier to which the liquid ismade to adhere.

According to the invention, a solution of a target substance that can bespecifically bonded to a probe is supplied to the probe carrier and putthem under appropriate reaction conditions to encourage them to bebonded to each other. When solutions of different target substancesneeds to be supplied to respective individual spots, at least a solutioncontaining at least a target substance that is in a dissolved state andneeds to be bonded to a probe is supplied to each of the plurality ofspots on the probe carrier.

The target substance may also be applied onto the probe carrier by meansof a liquid ejection device. Such an arrangement is preferable because avery small amount of liquid can be supplied quantitatively and theindexes of the probe carrier can also be used for regulating thealignment of the substrate and the ejection head.

Now, a method of detecting the position of a target substance that isspecifically bonded to a probe fixed onto a probe carrier according tothe invention that is prepared in a manner as described above will bedescribed below.

For detecting the position of a target substance, it is preferable todetect fluorescent light emitted from the fluorescent labels that arerelated to the target substance specifically bonded to a probeparticularly when the carrier has a division wall and openings (wells).

For the purpose of alignment of a scanned image in a detectingoperation, it is possible to identify a probe emitting fluorescent lightout of the probes formed on the carrier by taking an image of the probeson the probe carrier by means of an imaging means such as a CCD camera,using a laser as light source, processing the obtained image by means ofan image processing device, recognizing the image and detecting thecoordinates of the positions of the fluorescent labels by referring tothe coordinates of the positions of the indexes. If the indexes are verysmall particularly in terms of the line width thereof, a CCD cameracomprising an area sensor having a large number of pixels and/orprovided with a plurality of area sensors may preferably be used for thepurpose of the alignment.

The number of steps necessary for a surface treatment including a stepof albumin treatment can be reduced for the purpose of simplification byusing a method that does not detect information on the unnecessarysubstances remaining on the black matrix when detecting a probe emittingfluorescent light. For instance, a method of irradiating a laser beamfrom the rear surface of the carrier to detect fluorescent light emittedfrom a fluorescent substance or a method of detecting fluorescent lightemitted from a fluorescent substrate from the rear surface of thecarrier may be used.

EXAMPLE 1

Black resist containing carbon black (“CK-A143B”: tradename, availablefrom FUJIFILM Arch Co., Ltd.) was applied onto a glass substrate(“1737”: tradename, available from Corning), exposed to light by meansof a UV aligner in a predetermined manner, developed by means of anaqueous solution of inorganic alkali and subjected to a post baketreatment in which it was heated in a clean oven at 220° C. for 60minutes to prepare a black matrix pattern (division wall) having a filmthickness of 2 μm and rectangular openings of 100 μm×200 μm. Along withthe black matrix pattern, two cross-shaped indexes having a transversalpart that was 30 μm wide and 150 μm long and a vertical part that wasalso 30 μm wide and 150 μm long were formed at two corners of aperipheral part of the chip (see FIG. 1).

Subsequently, fluorescent coloring matter rhodamine B was dissolved intoa solvent to be used for ejecting the coloring matter by a liquidejection head that contains glycerol by 7.5 wt %, urea by 7.5 wt %,thiodiglycol by 7.5 wt % and acetylene alcohol expressed by generalformula (VII) (e.g., Acetylenol EH: tradename, available from KawakenFine Chemicals)

by 1 wt %, to a concentration of 10 μM. The coloring matter solution wasinjected into every other liquid reservoirs that were linked to therespective nozzles of an ink-jet head. Fluorescent coloring matter aminoFITC (concentration 10 μM) that was turned into a solution was injectedinto the remaining reservoirs into which no rhodamine B solution had notbeen injected. Subsequently, an application alignment was conducted byreferring to the cross-shaped indexes. In the operation of applicationalignment, the two cross-shaped indexes were shot by a CCD camera, usinga laser as a light source, and the obtained image was processed andrecognized by an image processing device to detect the coordinates ofthe indexes. Then, the head and the carrier were positionally aligned byregulating the XYθ stage of the liquid ejection device on the basis ofthe outcome of the coordinate detecting operation. The solutions of thetwo coloring matters were ejected from the respective nozzles of theliquid ejection head and applied onto a glass substrate that had beencleansed. Each of the reservoirs and the nozzles had been cleansed withsaid solvent and the corresponding coloring matter solution in advanceand the residual liquid was suctioned by vacuum. This cleansingoperation was repeated appropriately. A (main) droplet had a volume of10 pl and occupied a circular area with a diameter of about 50 μm whenthe droplet (dot) was applied onto the carrier. The gap separating twoadjacent dots on the carrier was about 100 μm.

An objective lens with a magnification of 20 (plan apochromaticobjective lens) and fluorescent light filters (B-2A: for rhodamine B,B-2E/C: for amino FITC, tradenames, both are available from Nikon Corp.)were put to a Nikon fluorescent microscope ECLIPSE E800 (tradename,available from Nikon Corp.) and the applied coloring matter solutionswere observed through the microscope with a magnification of 200 to findthat the aqueous solutions had been applied properly without producingany color mixture. The dots were arranged in an orderly manner. Nobiased arrangement nor stains attributable to mist and/or other causeswere observed.

EXAMPLE 2 Evaluation of Color Mixture by Hybridization

A glass substrate was cleansed as in Example 1. Subsequently, an aqueoussolution containing an aminosilane coupling agent (KBM-603: tradename,compound I, available from Shinetsu Chemical Co., Ltd.) expressed bychemical formula (I) of(CH₃O)₃SiC₃H₆NHC₂H₄NH₂  (I)that had been refined by distillation under reduced pressure to aconcentration of 1% was stirred at room temperature for an hour tohydrolyze the part of the methoxy group. Then, the substrate was dippedinto the aqueous solution of the silane coupling agent immediately afterthe cleansing at room temperature for an hour. Subsequently, thesubstrate was washed with flowing water (ultrapure water) and dried byblowing nitrogen gas. Then, it was heated to fix the coupling agent at120° C. in an oven.

After cooling the substrate, it was immersed into a 0.3% solution(ethanol:dimethylsulfoxide=1:1) of N-(6-maleimidecaproxy)succinimide(EMCS: compound II)

for two hours to cause EMCS to react with the amino group of theaminosilane coupling agent. After the reaction, the substrate was washedwith ethanol dimethylsulfoxide=1:1 once and with ethanol three times anddried by blowing nitrogen gas. 5′-ATGAACCGGAGGCCCATC-3′ SEQ13′-TACTTGGCCTCCGGGTAG-5′ SEQ2 5′-AAAAAAAAAAAAAAAAAAAAAAAAA-3′ SEQ33′-TTTTTTTTTTTTTTTTTTTTTTTTT-5′ SEQ4

Oligonucleotides (compounds III and IV: BEX Co. Ltd.) respectivelyhaving the base sequences of SEQ2 and SEQ4 that are complementary to thebase sequences of SEQ1 and SEQ3 and also at the 5′ end a mercapto group(SH group: also referred to as sulfidryl group) that can ultimatelyreact with and become bonded to a refined maleimide group on the surfaceof the substrate by way of a linker were utilized for oligonucleotideprobes. (III) 3′-TACTTGGCCTCCGGGTAG-OP(O₂)O—(CH₂)₆-5′ (IV)3′-TTTTTTTTTTTTTTTTTTTTTTTTT-OP(O₂)O—(CH₂)₆-5′

As in Example 1, each of the compounds III and IV was dissolved into asolvent to be used for ejection until the solutions showed a lightabsorbance of 1.0. Then, the obtained solutions were applied from aliquid ejection head onto the glass substrate to form spots that werearranged alternately as in Example 1. Each of the reservoirs and thenozzles had been cleansed with said solvent and the correspondingoligonucleotide solution in advance and the residual liquid wassuctioned by vacuum repeatedly. The volume of each liquid droplet, thedot size, the gap separating two adjacent dots, the ejection pattern,the number of times of ejection, the charging by means of an ionizer andthe charging of a mist adsorbing section were same as those ofExample 1. The solvent of this example has a high humidstatic effect andhence can prevent the inside of the reservoirs and the oligonucleotidesolutions on the substrate from drying.

The substrate onto which the solutions of the compounds III and IV hadbeen ejected was then put to react in a humidstat chamber showing ahumidity of 100% at room temperature for an hour and cleansed in flowingwater (ultrapure water) for about 30 seconds. Subsequently, thesubstrate was immersed in a 50 mM phosphate buffer solution (pH=7.0,containing 1M NaCl) containing BSA (bovine serum albumin, available fromSigma Aldrich Japan) to a concentration of 2% for an hour. Then, thesubstrate was appropriately washed with the buffer solution and storedin the latter.

(Hybridization)

As model target DNAs, compounds V and VI (purchased from BEX Co. Ltd.)having respectively the sequences of SEQ1 and SEQ3 of this example, tothe 5′ end of which tetramethylrhodamine was bonded, were used forhybridization.

As model target DNAs, compounds (V, VI): (only V is shown, the DNA partof VI is A25) having respectively DNA molecules and the sequences ofabove SEQ1 and SEQ3, to the 5′ end of which tetramethylrhodamine asfluorescent label was bonded,

were used and subjected to respective hybridization reactions with theprepared probes on the substrate.

The hybridization reactions were conducted in separate hybri-packs,using respectively two substrates and 2 ml of phosphate buffer solutions(10 mM phosphate buffer solutions, pH=7.0, containing 50 mM NaCl)containing the compounds V and VI to a concentration of 5 nM. The twosubstrates were put into the respective hybri-packs with the modeltarget DNA solutions and the hybri-packs were hermetically sealed. Theywere then heated to 70° C. in a humidstat tank and subsequently cooledto 50° C. Thereafter, they were left in that condition for 10 hours.

Then, the substrates were taken out from the hybri-packs and cleansedwith a buffer solution for hybridization for the purpose of removing theunreacted target DNAs. After the cleansing operation, the substratescovered by the buffer solution were put on a slide glass and covered bya cover glass. Then, fluorescent light from the fluorescent labels wereobserved by irradiating the substrates with a laser beam from the backsurface. The microscope used for the observation was a fluorescentmicroscope ECLIPSE E800 (tradename, available from Nikon Corp) providedwith an objective lens with a magnification of 20 (plan apochromat) anda fluorescent light filter (Y-2E/C). Fluorescent light was observed onlyfrom the parts of each of the substrates where probes complementary tothe target DNA existed. The dots of the probes were found to be arrangedin an orderly manner.

EXAMPLE 3

[Formation of Black Matrix]

Black resist (CK-A143B Resist: tradename, available from FUJIFILM ArchCo., Ltd.) containing carbon black was applied onto a glass substrate(1737: tradename, available from Corning) and subjected to apredetermined exposure session using a UV aligner and a developingoperation using an inorganic aqueous alkali solution. Then, thesubstrate was heated in a clean oven at 220° C. for 60 minutes andsubjected to a post bake treatment to prepare a black matrix pattern(division wall) having a film thickness of 2 μm and rectangular openingsof 100 μm×200 μm. The length a of the division wall was made equal to 20μm.

[Evaluation of Surface Coarseness]

The surface coarseness of the glass substrate used for forming the blackmatrix was observed at arbitrarily selected areas by using NanoScopeIIIa AFM Dimension 3000 Stage System (tradename, available from DigitalInstrument) before forming the black matrix. As a result, the substrateshowed an average surface coarseness (Ra) of 3 nm.

[Dry Etching Treatment]

The glass substrate on which a black matrix had been formed (blackmatrix substrate) was subjected to a plasma treatment under thefollowing conditions, using a parallel plates type plasma treatmentapparatus that employs the cathode coupling system. gas:   o₂ gas flowrate:   80 sccm pressure:   8 Pa RF power: 0.15 KW treatment time:   30sec[Plasma Treatment]

After the completion of the dry etching treatment, the black matrixsubstrate was subjected to a plasma treatment under the followingconditions, using the same apparatus. gas: CF₄ gas flow rate: 330 sccmpressure:  44 Pa RF power:  1.2 KW treatment time: 130 sec[Water Treatment]

The black matrix substrate that had been subjected to a plasma treatmentwas then subjected to a water treatment. More specifically, the blackmatrix substrate was immersed into a ultrasonic wave pure water tankunder the following conditions. pure water temperature: 50 to 60° C.ultrasonic wave frequency: 40 kHz treatment time:  5 min drying: liftingand drying, 60° C., 5 min[Evaluation of Water Repellency]

The black matrix substrate that had been subjected down to the watertreatment was observed for the contact angle relative to pure water bymeans of an automatic liquid crystal glass cleaning/treatment inspectionapparatus “LCD-400S” (tradename, available from KYOWA INTERFACE SCIENCECO., Ltd.). The surface of the black matrix was observed on a framehaving a width of 5 mm and arranged around the micro pattern, whereasthe surface of the glass substrate was observed in an area outside ofthe frame where no black matrix pattern was formed. The contact anglesof the surfaces were as follows. glass substrate surface:  12° blackmatrix surface: 125°[Supply of Aqueous Solution to Carrier by Means of Ink-Jet System]

Subsequently, fluorescent coloring matter rhodamine B was dissolved inan aqueous solution containing the solvent to be ejected from a liquidejection head that contained glycerol by 7.5 wt %, urea by 7.5 wt %,thiodiglycol by 7.5 wt % and acetylene alcohol expressed by generalformula (VII) (e.g., Acetylenol EH: tradename, available from KawakenFine Chemicals)

by 1 wt % to a concentration of 10 μM. Subsequently, the rhodamine Baqueous solution was supplied to each opening by 20 pl by means of anink-jet apparatus. Subsequently, a 10 μM aqueous solution of amino FITCwas supplied by 20 pl to another first region by means of anotherink-jet head. The use of rhodamine B and amino FITC was selected becausethey dissolve in water and are advantageous for observing the state ofthe supplied liquid and that of cross contamination by means offluorescent light.

The contact angle of the above black matrix substrate relative to theprobe solution was observed by means of an automatic liquid crystalglass cleaning/treatment inspection apparatus “LCD-400S” (tradename,available from KYOWA INTERFACE SCIENCE CO., Ltd.). The surface of theblack matrix was observed on a frame having a width of 5 mm and arrangedaround the micro pattern, whereas the surface of the glass substrate wasobserved in an area outside of the frame where no black matrix patternwas formed. The contact angles of the surfaces were as follows. glasssubstrate surface:  17° black matrix surface: 115°

A G excitation filter (for rhodamine B) and a B excitation filter (foramino FITC) were fitted to a Nikon fluorescent microscope and each ofthe supplied aqueous solutions was observed for its state by usingfluorescent light. It was found that each aqueous solution had beensupplied uniformly in the first region without forming any liquiddroplet. It was also found that no fluorescent light of any foreigncoloring matter was observed in the first region where the propercoloring matter was to be supplied. Therefore, it was found that anaqueous solution can be supplied to each first region without crosscontamination. When the diameter of the applied liquid droplet wasobserved, it was found that the solution had satisfactorily wetted thesubstrate and spread in the opening so that the edges of the liquiddroplet could not be detected. No blank area was observed in all thespecimens of probe carrier prepared in this example.

COMPARATIVE EXAMPLES 1 AND 2

A probe carrier was prepared as in Example 3 except that the plasmatreatment and the water treatment were not conducted. The contact anglesof the black matrix substrate relative to pure water were as follows.glass substrate surface: 80° black matrix surface: 80°

A probe carrier was prepared as in Example 1 except that no watertreatment was conducted. The contact angles of the black matrixsubstrate relative to pure water were as follows. glass substratesurface:  15° black matrix surface: 130°

The spreading property of the solution was also observed to find thatthe probe solution of an amount of 20 pl had not wetted the substratenor spread in the opening and a blank area was observed in all theopenings.

EXAMPLE 4

A probe carrier was prepared in a manner as described above by referringto Example 1 and a dry etching treatment, a plasma treat and a watertreatment were also conducted as pretreatment for applying probesolutions onto the carrier as in Example 3.

An objective lens with a magnification of 20 (plan apochromaticobjective lens) and fluorescent light filters (B-2A: for rhodamine B,B-2E/C: for amino FITC, tradenames, both are available from Nikon Corp.)were put to a Nikon fluorescent microscope ECLIPSE E800 (tradename,available from Nikon Corp) and the applied coloring matter solutionswere observed through the microscope with a magnification of 200 to findthat the aqueous solutions had been applied properly without producingany color mixture. The dots were arranged in an orderly manner. Nobiased arrangement nor stains attributable to mist and/or other causeswere observed. Furthermore, it was found that the solution hadsatisfactorily wetted the substrate and spread in the opening so thatthe edges of the liquid droplet could not be detected. No blank area wasobserved in all the specimens of probe carrier prepared in this example.

In this example, the probe carrier was provided with indexes andsubjected to a plasma treatment and a de-fluorine treatment. In otherwords, a carrier having a division wall that was highly repellentrelative to aqueous probe solutions and probe fixing regions that werehighly hydrophilic was prepared. Then, the probe solutions of adjacentopenings (well) of the division wall were never mixed with each otherand the intended probe solutions could be applied to the intendedrespective positions if some of the probe solutions were displacedslightly from the respective proper positions where they were to beapplied when an operation of application alignment was conducted byreferring to the indexes.

Thus, according to the invention, it is possible to accurately, quicklyand reliably manufacture a probe carrier that densely carries probes byusing a carrier having indexes in a probe non-fixing region, applyingsolutions containing probes to specific positions on the carrier byreferring to the indexes and fixing the probe. Additionally, accordingto the invention, it is possible to accurately and quickly detect theposition of the target substance that is specifically bonded to a probefixed onto the probe carrier manufactured by a method according to theinvention by referring to the indexes.

1. A method of manufacturing a probe carrier carrying probes of aplurality of species fixed at respective specific different positions onthe carrier and adapted to be specifically bonded to a target substance,said method comprising: a step of forming one or more than one labeledindexes on said carrier at a position out of said specific positions,wherein each labeled index has a label that produces radiation underpredetermined conditions; and a step of applying solutions respectivelycontaining said probes to said respective specific positions byreferring to said indexes.
 2. A method according to claim 1, whereinsaid carrier has a division wall for partitioning said specificpositions, and wherein said indexes are located at predeterminedpositions of said division wall.
 3. A method of manufacturing a probecarrier carrying probes of a plurality of species fixed at respectivespecific positions on the carrier and adapted to be specifically bondedto a target substance, said method comprising: a step of forming adivision wall for partitioning said specific positions on said carrier;a plasma irradiation step of irradiating said carrier with plasma in agas atmosphere containing at least fluorine atoms; and a step ofremoving any ingredients of said gas adhering to said specificpositions.
 4. A method according to claim 3, wherein said removing stepis a washing step of washing and removing any ingredients of said gaswith water by bringing water into contact with the surface of thecarrier subjected to said plasma treatment.
 5. A method according toclaim 3, wherein said division wall is formed by a photolithographymethod.
 6. A method according to claim 5, wherein said photolithographymethod includes: a step of forming said division wall by forming aphotosensitive resin layer on the surface of said carrier, exposing saidphotosensitive resin layer to light to form a pattern corresponding tosaid division wall, and developing said photosensitive resin layer; anda step of further baking said division wall prior to said plasmairradiation step.
 7. A method according to claim 4, wherein, afterbringing said carrier into contact with water in said washing step, saidcarrier is dried at a temperature not exceeding 100° C.
 8. A methodaccording to claim 1, wherein said step of applying solutions containingsaid probes to said respective specific positions is a step of applyingsaid solutions by causing them to fly in the air.
 9. A method ofmanufacturing a probe fixing carrier for carrying probes of a pluralityof species fixed at respective specific positions on the carrier andadapted to be specifically bonded to a target substance, said methodcomprising: a step of forming a division wall for partitioning regionson said carrier; a plasma irradiation step of irradiating said carrierwith plasma in a gas atmosphere containing at least fluorine atoms; anda washing step of washing and removing any ingredients of said gas withwater by bringing water into contact with the surface of the carriersubjected to said plasma treatment.
 10. A method of manufacturing aprobe carrier, said method comprising: applying solutions containingprobes to respective specific positions on a probe carrier manufacturedby a method according to claim 9 and fixing said probes.
 11. A probecarrier carrying probes of a plurality of species fixed at respectivespecific different positions on the carrier at probe fixing regions andadapted to be specifically bonded to a target substance, said probecarrier comprising: one or more than one indexes formed on said carrierat positions out of said specific positions; wherein said carrier has adivision wall for partitioning said specific positions, and wherein saidindexes are located at predetermined positions of said division wall;wherein said division wall has water repellency and the contact angle ofsaid division wall relative to pure water is not less than 120°; andwherein said probe fixing regions are hydrophilic and the contact angleof said probe fixing regions relative to pure water is not more than30°.
 12. (canceled)
 13. A probe carrier according to claim 11, whereinsaid indexes are recesses formed in said division wall.
 14. A probecarrier according to claim 11, wherein said division wall has alight-shielding effect.
 15. A probe carrier according to claim 11,wherein said division wall is made of a resin composition containingcarbon black.
 16. (canceled)
 17. (canceled)
 18. A probe fixing carrierfor carrying probes of a plurality of species fixed at respectivespecific different positions on the carrier at probe fixing regions andadapted to be specifically bonded to a target substance, said probefixing carrier comprising: one or more than one indexes formed on saidcarrier at positions out of said specific positions; wherein saidcarrier has a division wall for partitioning said specific positions,and wherein said indexes are located at predetermined positions of saiddivision wall; wherein said division wall has water repellency and thecontact anile of said division wall relative to pure water is not lessthan 120°; and wherein said probe fixing regions are hydrophilic and thecontact angle of said probe fixing regions relative to pure water is notmore than 30°.
 19. (canceled)
 20. A probe fixing carrier according toclaim 18, wherein said indexes are recesses formed in said divisionwall.
 21. A probe fixing carrier according to claim 18, wherein saiddivision wall has a light-shielding effect.
 22. A probe fixing carrieraccording to claim 18, wherein said division wall is made of a resincomposition containing carbon black.
 23. (canceled)
 24. (canceled)
 25. Amethod of locating a position of a probe specifically bonded to a targetsubstance out of a number of probes of a plurality of species by causingsaid probes of a plurality of species carried on the carrier to contactthe target substance, said method comprising: a step of locating theposition of the probe specifically bonded to the target substance byreferring to indexes formed at positions out of the specific positionscarrying said probes of a plurality of species; wherein said indexes andsaid target substance have a label that produces radiation underpredetermined conditions.
 26. (canceled)
 27. A method according to claim25, wherein said label is a fluorescent substance.
 28. A methodaccording to claim 27, wherein said carrier is a light-transmittingplate-shaped body and the predetermined positions for fixing said probesare partitioned by a light-shielding division wall arranged on one ofthe surfaces of said carrier, information on the position of said targetsubstance being obtained by detecting fluorescent light emitted fromsaid label and passing through said carrier at the opposite surface ofsaid carrier.